To the Editor: Candidatus Neoehrlichia mikurensis is a member of the order Rickettsiales, family Anaplasmataceae (1). Manifestations of infection with these bacteria are atypical and severe and include cough, nausea, vomiting, anemia, headache, pulmonary infiltration, malaise, myalgia, arthralgia, fatigue, recurrent fever for ≤8 months, and/or death (2–5). Candidatus N. mikurensis has been detected in Ixodes ovatus, I. persulcatus, and Haemaphysalis concinna ticks in Asia (1,5).

Candidatus N. mikurensis has been identified as one of the most prevalent pathogenic agents in I. ricinus ticks throughout Europe (2,3,6). Rodents of diverse species and geographic origins have been shown to carry these bacteria, but transmission experiments have not been conducted to unambiguously identify natural vertebrate reservoirs (1–3,5–7). This emerging tickborne pathogen has been detected mainly in immunocompromised patients in Sweden (n = 1), Switzerland (n = 3), Germany (n = 2), and the Czech Republic (n = 2) and in immunocompetent patients in China (n = 7) (2–5).

Anaplasma phagocytophilum is an obligate, intracellular, tickborne bacterium of the family Anaplasmataceae and causes granulocytic anaplasmosis in humans and domestic animals. In Europe, I. ricinus ticks are its major vector, and red deer, roe deer, rodents, and European hedgehogs (Erinaceus europaeus) are suspected reservoir hosts (8).

Northern white-breasted hedgehogs (Erinaceus roumanicus) are urban-dwelling mammals (order Eulipotyphla, family Erinaceidae) that serve as major maintenance hosts for the 3 stages of I. ricinus ticks (9). However, E. roumanicus hedgehogs have not been studied for their ability to carry A. phagocytophilum. In addition, no suspected reservoirs other than rodents have been investigated for Candidatus N. mikurensis. The purpose of this study was to determine whether this hedgehog is a potential reservoir of these 2 bacteria.

We conducted an ecoepidemiologic study during 2009–2011 to obtain information about ticks and tickborne pathogens of urban hedgehogs in a park on Margaret Island in central Budapest, Hungary (9). Ear tissue samples were obtained from hedgehogs anesthetized with intramuscular ketamine (5 mg/kg) and dexmedetomidine (50 µg/kg).

Candidatus N. mikurensis was detected in 2 (2.3%) of 88 hedgehog tissue samples. Formerly, rodents were the only wild mammals found to act as potential reservoirs for this pathogen. Results of studies that attempted to detect these bacteria in common shrews (Sorex araneus), greater white-toothed shrews (Crocidura russula) (2,3), or common moles (Talpa europaea) (2) were negative. However, our results indicate that northern white-breasted hedgehogs might be a non-rodent reservoir for Candidatus N. mikurensis.

The low pathogen prevalence observed in this urban hedgehog population compared with that in rodents in other locations (2,3) might be caused by use of skin samples. Skin samples from rodents showed only 1.1% positivity in a study in Germany; however, average prevalence of Candidatus N. mikurensis in transudate, spleen, kidney, and liver samples from the same animals was 37.8%–51.1% (2). Although we did not test other organs, we hypothesize that prevalence of Candidatus N. mikurensis infection urban hedgehogs is probably >2.3%.

We detected A. phagocytophilum in 67 (76.1%) of 88 urban hedgehogs. This prevalence was similar to that found among European hedgehogs in Germany (8). I. ricinus ticks are more common than I. hexagonus ticks in this urban hedgehog population (9). Thus, I. ricinus ticks can acquire these bacteria when feeding on hedgehogs and the risk for human infection with A. phagocytophilum in this park in Budapest is relatively high.

Neoehrlichiosis and granulocytic anaplasmosis have not been diagnosed in humans in Hungary. This finding is probably caused by diagnostic difficulties rather than absence of these pathogens in the environment. Infection with Candidatus N. mikurensis and A. phagocytophilum cause predominantly noncharacteristic symptoms. Laboratory cultivation and serologic detection of Candidatus N. mikurensis has not been successful, and this pathogen has not been identified in blood smears. Thus, accurate diagnosis of suspected cases requires suitable molecular methods.

Parks can be considered points of contact for reservoir animals, pathogens, ticks, and humans. Our results indicate that E. roumanicus hedgehogs play a role in urban ecoepidemiology of ≥2 emerging human pathogens. To better understand the urban cycle of these pathogens, potential reservoir hosts, ticks collected from these hosts, and vegetation in parks should be investigated.

We thank the Middle Danube Valley Inspectorate for Environmental Protection, Nature Conservation and Water Management, Hungary, for approving capturing and anesthetizing of hedgehogs and sample collection.

This study was partially supported by European Union grant FP7-261504 EDENext and was cataloged by the EDENext Steering Committee as EDENext148 (www.ede.next.euExternal Web Site Icon). G.F. was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences and an NKB grant from the Faculty of Veterinary Science, Szent István University. E.C.C. and H.S. were supported by EurNegVec Cost Action TD1303.

‘Candidatus Neoehrlichia mikurensis’ is a tick-borne bacteria implicated in human health. To date, ‘Ca. Neoehrlichia mikurensis’ has been described in different countries from Africa, Asia and Europe, but never in Spain. However, according to the epidemiological features of the main vector in Europe, Ixodes ricinus, its circulation in our country was suspected.

Methods

A total of 200 I. ricinus ticks collected in the North of Spain were analyzed. DNAs were extracted and used as templates for PCRs targeting fragment genes for Anaplasma/Ehrlichia detection. The amplified products were sequenced and analyzed.

The detection of ‘Ca. Neoehrlichia mikurensis’ in I. ricinus ticks from Spain indicates its circulation and the potential risk of contracting a human infection in this country. Keywords: ‘Candidatus Neoehrlichia mikurensis’; Anaplasma phagocytophilum; Ixodes ricinus; Spain

Background

‘Candidatus Neoehrlichia mikurensis’ is an obligate intracellular bacterium member of the Anaplasmataceae family. It was first isolated from wild rats (Rattus norvegicus) and Ixodes ovatus ticks in the Mikura Island, Japan [1]. It was classified as a new genus (Neoehrlichia) added to those already known of the Anaplasmataceae family: Ehrlichia, Anaplasma, Neorickettsia, Aegyptianella and Wolbachia[1].

The presence of ‘Ca. Neoehrlichia mikurensis’ in rodents and ticks has been notified from different countries of Europe, Asia and Africa in the last decade [2,3]. In Europe, it has been mostly detected in Ixodes ricinus, although it has been associated to other tick species in other continents. I. ricinus, endemic in the North of Spain, is responsible for most human tick bites. It acts as vector of different human pathogens, such as Borrelia burgdorferi sensu lato (s.l.), Anaplasma phagocytophilum -formerly Ehrlichia phagocytophila- or different Rickettsia spp., protozoa and arboviruses. However, the risk of infections with ‘Ca. Neoehrlichia mikurensis’ to human health remains unclear in southern Europe.

The first implication of the bacterium in human pathology was reported in Sweden in 2010 [4]. Subsequently, seven new human cases severely affected by ‘Ca. Neoehrlichia mikurensis’ infections have been notified from Europe [5-8]. Several human cases have also been described in China [2].

‘Ca. Neoehrlichia mikurensis’ has not been previously described in Spain. However, according to the epidemiological features of the main vector, I. ricinus, in which the bacterium has been mostly detected in Europe, its circulation in our country was suspected.

Methods

In the routine analysis of tick-borne pathogens performed in the Center of Rickettsioses and Arthropod-borne Diseases (Logroño, Spain), 200 I. ricinus ticks collected from cows were tested. Samples were obtained in two different locations of La Rioja (Spain): Tobía (42°18’N; 2°48’W) and Jubera (42°18’N; 2°17’W) in April 2013. A total of 50 males and 50 engorged females from each location were processed. Ticks were kept at -80°C until DNA extraction with Qiagen DNeasy Blood & Tissue Kit, according to the manufacturer’s instructions (Qiagen, Hilden, Germany).

All DNA extracts were used as templates for two nested PCRs targeting fragment genes for Anaplasma/Ehrlichia detection. Furthermore, a simple PCR was performed to confirm the amplification of species never detected in the area (Table 1) [9-11]. Two negative controls, one of them containing water instead of template DNA and the other with template DNA but without primers, as well as a positive control of A. phagocytophilum were included in all PCR assays. Amplification products were sequenced, and nucleotide sequences were compared with those available in GenBank by using a Basic Local Alignment Search Tool (BLAST) search (http://www.ncbi.nlm.nih.gov/blast webcite).

Table 1. PCR primer pairs used in this study

Results and discussion

Two nucleotide sequences of groESL fragment gene (1%) corresponding to 2 male tick specimens collected in Tobía showed 100% identity with ‘Ca. Neoehrlichia mikurensis’. They were identical to the one detected in two patients in Germany [5]. None of them yielded positive results when PCR tests for 16S rRNA were performed. For this reason, a different fragment of the 16S rRNA gene (EHR) was investigated to confirm our previous results. Nucleotide sequences of both samples were identical to each other and showed 100% identity with more than one sequence of ‘Ca. Neoehrlichia mikurensis’ (Table 2). In our laboratory we had never worked with ‘Ca. Neoehrlichia mikurensis’ before, so no contamination with this bacterium was possible.

On the other hand, A. phagocytophilum was detected in 61 samples (30.5%) of this study. Specifically, 8 specimens (4%) showed maximum identity with the human pathogenic variant, and 53 (26.5%) with non-pathogenic variants (Table 2).

In this study, ‘Ca. Neoehrlichia mikurensis’ DNA was detected in two ticks from La Rioja (Spain) during 2013 but we do not know if this bacterium has been previously circulating in our area. Anyway, this infection may be underdiagnosed in our media. In addition, according to the recent finding of several human cases due to this bacterium, mainly in immunocompromised patients, physicians should be aware of the risk for those patients in the affected area. Moreover, infections and fever of unknown origin are common in immunocompromised patients and the responsible pathogen is not isolated in most cases [7]. The detection of ‘Ca. Neoehrlichia mikurensis’ and the features of the European human cases suggest that this microorganism is likely causing disease in our country too.

The prevalence of A. phagocytophilum in the studied area has been previously reported [12]. According to our results, the high prevalence of the bacterium in the engorged females collected in Jubera should be noted (40 out of 50 specimens, 80%). This could be due to the fact that all the female specimens processed were engorged on cows, hosts that are potential amplifiers of the bacterium [13].

Conclusions

‘Ca. Neoehrlichia mikurensis’ has been detected in I. ricinus ticks removed from cows in Spain. A. phagocytophilum was amplified in 61 out of 200 samples (8 of them corresponding to the human pathogenic variant). Our results suggest that human infections by ‘Ca. Neoehrlichia mikurensis’ might be undiagnosed in this country. Further research should be carried out to study the epidemiology of the bacterium as well as to be aware of possible human cases.

This is the first reporting of the tick-borne zoonotic bacterium “Candidatus Neoehrlichia mikurensis” in Denmark. A total of 2,625 Ixodes ricinus ticks from 58 locations in Denmark were collected and analysed for “Ca. Neoehrlichia mikurensis”. A nested PCR revealed the presence of the bacterium at three geographically separate locations, which indicates that it is widely established in ticks.

Background

Since 2009, “Candidatus Neoehrlichia mikurensis” has been correlated with severe disease in immunocompromised people. A total of six human cases originating from Sweden [1], Germany [2], Switzerland [3] and the Czech Republic [4] have been described in the literature. In 2011, the bacterium was also isolated from a post-operative dog in Germany [5]. General clinical features in human patients have included recurrent fever, erysipelas-like rashes, arthralgias and thromboembolisms [1-4]. The infection responds well to doxycycline [1]. The pathogenic potential of “Ca. Neoehrlichia mikurensis” may be correlated with its putative tropism for endothelial cells [2,4].

So far little is known on the distribution, risk areas and reservoir of “Ca. Neoehrlichia mikurensis”. If the infectious cycle resembles the other Ehrlichia bacteria, it has its reservoir in wild mammals and is transmitted by ticks. Accidentally humans may become infected [6]. In this study we examined Ixodes ricinus ticks in Denmark for the presence of “Ca. Neoehrlichia mikurensis” using PCR. This is the first survey for “Ca. Neoehrlichia mikurensis” in Denmark and in ticks in northern Europe.

Sampling methods and analysis

The analysed ticks originated from two different sampling procedures (Table): ticks collected by flagging (n=1,552) and a tick DNA/RNA archive (n=1,073).

Table. Ticks collected by two different sampling procedures in Denmark in 2010 and 2011 (n=2,625)

Flagging was performed during September 2011 at four distinct localities known for an abundance of ticks and a recent history of human cases of tick-borne encephalitis (TBE). A white flannel flag was dragged over the vegetation and 1,552 ticks collected into plastic containers. These were frozen a few hours after collection and stored at -20˚C for up to one month before DNA extraction. The flagging for ticks was carried out as part of a project investigating TBE virus. However, with the emergence of “Ca. Neoehrlichia mikurensis” as a public concern in our neighbouring countries, the DNA was additionally screened for the presence of this potentially emerging pathogen in December 2011 and January 2012.

Furthermore, a tick archive was investigated for the presence of “Ca. Neoehrlichia mikurensis”. During 2010 and 2011, the Veterinary Institute’s National Center for Wildlife Health collected 1,073 ticks from roe deer submitted for diagnosis and routine surveillance from 53 locations in Denmark. A sample of 40 ticks from a domestic sheep flock was additionally included in the archive. After removal, ticks were stored in ethanol for up to 1.5 years. DNA and RNA were extracted and stored as a tick archive of genetic material.

Before laboratory analysis, ticks from sites with large sample sizes were distributed into smaller pools (Table). Ticks were crushed and homogenised in 1 ml phosphate buffered saline (PBS). The homogenate was centrifuged and supernatant collected and stored at -80˚C until DNA was extracted from 200 µL homogenate in a MagNA Pure 96 robot using MagNa Pure 96 DNA and Viral Nucleic Acid Small Volume Kit version 4.0 (Roche) according to the manufacturer’s instructions.

Three of the 79 pools contained ticks positive for “Ca. Neoehrlichia mikurensis”; they originated from three locations separated from each other by the sea (Figure).

Figure. Location of tick collection, Denmark, 2010 and 2011 (n=1,552)

The first positive sample came from flagging in Øster Sømarken on the island of Bornholm. From this location six pools with a total of 467 ticks were collected. One pool, containing 100 nymphs, was found positive for “Ca. Neoehrlichia mikurensis”. The second positive pool was found after flagging in the forest of Tokkekøb Hegn in Northern Zealand in the same one-hectare area where emerging TBE in both I. ricinus and humans were reported in 2009 [8]. At this location eight pools with a total of 736 ticks were collected, of which one, containing 100 nymphs, was found positive. The third positive sample originated from a pool of 12 male and 28 female ticks collected for the tick archive from domestic free-grazing sheep in the area of Viborg on the Danish mainland. All three isolates were verified to be 100% similar to 16S rRNA gene sequences from “Ca. Neoehrlichia mikurensis”.

Discussion

“Ca. Neoehrlichia mikurensis” belongs to the family Anaplasmataceae [9] which comprises a variety of emerging tick-borne human pathogenic bacteria [10]. Former studies have suggested a potential widespread occurrence of “Ca. Neoehrlichia mikurensis” in the wild fauna of Asia and Europe, including our neighbouring countries [9,11-14], but it has never been reported in Denmark. In this study we examined 2,625 I. ricinus ticks divided in 79 pools and identified the presence of “Ca. Neoehrlichia mikurensis” at three distinct locations, indicating that the bacterium is widely distributed in the Danish tick population.

The recorded minimum prevalence of three of 2,625 was, however, substantially lower than that found in studies from the Netherlands in 2006, the Baltic regions of Russia in 1997–98 and a recent central European study, which all estimated 6–7% of the ticks to carry the bacterium [12,14,15]. The latter study investigated ticks from the Czech Republic, France, Germany, Poland and Portugal and found a prevalence ranging from 0% to 10% that was highest in the Czech Republic and Germany [14]. In southern Sweden, “Ca. Neoehrlichia mikurensis” was in 2008 found to be widespread in the wild rodents of this region with a prevalence ranging from 0% to 12.5% in the investigated locations [11].

An increase and spread of other tick-borne infections such as Lyme borreliosis and TBE, has been reported in Denmark and neighbouring countries. This trend has been attributed to increased awareness, climate change and increasing tick populations [16,17]. In this study the tick-borne pathogen was found at a known TBE site on Bornholm and at an emerging TBE site in Tokkekøb Hegn forest [8]. The recent appearance of several human clinical cases of “Ca. Neoehrlichia mikurensis” infection in Europe, as well as the findings in the wild fauna, indicate that this is an emerging tick-borne pathogen. However, lack of knowledge and a diagnostic test combined with a low pathogenic potential may have hindered previous detection in Denmark. Whether or not the newly reported cases are the result of previous misdiagnosis or a true emerging risk, it is important that medical doctors in the affected areas are aware of the risk for immunocompromised patients. The State Serum Institute will now establish a diagnostic assay. Finding the pathogen on production animals suggests there may be veterinary risk as well.

AcknowledgementsTick-sampling was funded by Baxter, while laboratory work was carried out as part of a two-year collaboration on tick-borne pathogen detection between the Club5 institutes ANSES in France, SVA in Sweden, CVI in the Netherlands and DTU in Denmark 2011–12. Anne Lyhning Jensen and Christian Fertner are acknowledged for skilful technical assistance.